Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy

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Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force

microscopy

Cécile Formosa-Dague, Marion Schiavone, Anita Boisrame, Mathias Richard, Raphael Duval, Etienne Dague

To cite this version:

Cécile Formosa-Dague, Marion Schiavone, Anita Boisrame, Mathias Richard, Raphael Duval, et al..

Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy. Nanomedicine: Nanotechnology, Biology and Medicine, Elsevier, 2015, 11 (1), pp.57-65.

�10.1016/j.nano.2014.07.008�. �hal-01553144�

Multiparametric imaging of adhesive nanodomains at the surface of

1

Candida albicans by Atomic Force Microscopy

2 3

Cécile Formosa, MS

, Marion Schiavone, MS

Anita Boisrame, PhD

, Mathias L. Richard, 4

PhD

, Raphaël E. Duval, PhD

, and Etienne Dague, PhD

5

6

1

CNRS, LAAS, 7 avenue du Colonel Roche, F-31400 Toulouse, France 7

2

Université de Toulouse ; LAAS, F31400 Toulouse, France 8

3

CNRS, UMR 7565, SRSMC, Vandœuvre-lès-Nancy, France 9

4

Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France 10

5

INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France 11

6

AgroParisTech, UMR Micalis, F-78850 Thiverval Grignon, France 12

7

ABC Platform®, Nancy, France 13

14

Corresponding author: Etienne Dague, LAAS-CNRS, 7 av du Colonel Roche, 31400 Toulouse, 15

France. Phone number: +33 5 61 33 78 41 Mail : edague@laas.fr 16

17

Abstract word count: 138 words / Manuscript word count: 4451 words / Number of figures: 5 / 18

Number of references: 40 19

20

This work was supported by an ANR young scientist program (AFMYST project ANR-11-JSV5- 21

001-01 n° SD 30024331) to ED. CF and MS are respectively supported by a grant from 22

“Direction Générale de l’Armement” (DGA) and from Lallemand SAS.

23

24

Abstract 25

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety 26

of adhesins that interact with hosts ligands. The spatial organization of these adhesins on the 27

cellular interface is however poorly understood, mainly because of the lack of an instrument able 28

to track single molecules on single cells. In this context, the Atomic Force Microscope (AFM) 29

makes it possible to analyze the force signature of single proteins on single cells. The present 30

study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed 31

that the adhesins at the cell surface were organized in nanodomains composed of free or 32

aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and 33

synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids 34

nanodomains will help in understanding the virulence factors of C. albicans.

35

36

37

Background 38

The yeast Candida albicans has emerged as a major public health problem these last two 39

decades. This opportunistic pathogen causes a wide range of infections from surface infections, to 40

mucosal and blood-stream infections

. Whereas mucosal infections are common and occur in 41

healthy organisms, blood-stream infections are observed only in immunocompromised patients 42

and are life-threatening. This type of infections, also known as candidaemia, can develop into 43

disseminated candidiasis when the infection spreads to internal organs, leading to high mortality 44

rates

. In order to colonize and subsequently to disseminate in the blood stream C. albicans needs 45

to adhere to different substrates. This first stage of infection

is mediated by adhesins that are 46

found on the surface of the yeast cell wall. Many of these adhesins are mannoproteins, and 47

among them, the adhesin family identified as having a major role in host cell attachment is the 48

Als (Agglutinin-like Sequences) family

. 49

The Als were initially reported as having homologies with the proteins responsible for 50

auto-agglutination in the baker yeast Saccharomyces cerevisiae. Eight Als have been identified, 51

they all are primarily involved in host-pathogen interactions

. It was found that there were 52

amyloid-forming sequences in the Als adhesins of Candida albicans

. Amyloids are insoluble 53

fibrillar protein aggregates whose core consists in crystalline arrays of identical sequence in many 54

molecules of the amyloid protein

. Cells expressing the Als proteins can rapidly aggregate, and 55

the aggregation has amyloid-like properties. Like amyloid formation, aggregation ability 56

propagates through the adherent cell population and depends on conformational changes of the 57

Als protein. This transition of the conformational state to an aggregative state of the proteins is 58

characterized by the formation of hydrophobic nanodomains on the entire surface of the cell

. 59

A few papers written by Lipke’s team were dedicated to the direct visualization of these 60

nanodomains using fluorescent dyes such as thioflavin T or 8-anilino-1-naphtalene-sulfonic acid

61

(ANS)

. Another technique that can be used to visualize these nanodomains is Atomic Force 62

Microscopy (AFM). AFM has recently emerged as a valuable tool to study the surface of living 63

cells

, and especially pathogenic cells

. This technology has been used by Alsteens et al. to 64

image the formation and propagation of nanodomains in living yeast cells

and also to unfold 65

amyloid proteins from the yeasts surface using Single Molecule Force Spectroscopy

. To this 66

end, the authors functionalized AFM tips with antibodies targeted against the Als protein directly 67

or against an epitope tag present in the Als protein. These studies allowed the authors to localize 68

the adhesive nanodomains caused by the aggregation of Als proteins at the surface of living yeast 69

cells, and to unravel the structure of the Als proteins studied by stretching.

70

In our study, we used AFM as an imaging tool to visualize and localize adhesins 71

nanodomains at the surface of living wild-type Candida albicans cells. Using recent 72

developments in the AFM technology, we have imaged and quantified at the same time the 73

nanomechanical properties, the adhesiveness (force and nature of the interaction), the size and the 74

thickness of the nanodomains

, at high resolution. The data collected showed that these 75

nanodomains are localized differently at the surface of the cell, depending on the structures 76

featured by the cells (bud scars, buds). We also showed that there were degrees of adhesiveness, 77

depending on whether the amyloid proteins had totally aggregated (hydrophobic nanodomains) or 78

not, and that these degrees of aggregation were directly correlated to the stiffness of the yeast cell 79

wall. Finally, using force measurements and amyloid forming or inhibiting peptides, we showed 80

that Als proteins (probably among others) were participating to these nanodomains.

81 82

Methods 83

Yeasts growth conditions

84

Candida albicans (from ABC Platform® Bugs Bank, Nancy, France) was stocked at -80°C, 85

revivified on Yeast Peptone Dextrose agar (Difco, 242720-500g) and grown in Yeast Peptone 86

Dextrose broth (Difco, 242820-500g) for 20 hours at 30°C under static conditions.

87 88

Sample preparation for AFM experiments 89

Yeast cells were concentrated by centrifugation, washed two times in acetate buffer (18 mM 90

CH

COONa, 1 mM CaCl

, 1 mM MnCl

, pH = 5.2), resuspended in acetate buffer, and 91

immobilized on polydimethylsiloxane (PDMS) stamps prepared as described by Dague et al

. 92

Briefly, freshly oxygen activated microstructured PDMS stamps were covered by a total of 100 93

µL of the solution of cells and allowed to stand for 15 minutes at room temperature. The cells 94

were then deposited into the microstructures of the stamp by convective/capillary assembly.

95

Images were recorded in acetate buffer in Quantitative Imaging

mode with MLCT AUWH 96

(Bruker) cantilevers (nominal spring constant of 0.01 N/m). The applied force was kept at 1.5 nN 97

for imaging and at 0.5 nN for force spectroscopy experiments. The loading rate for imaging was 98

of 2 500 000 pN/s (acquisition frequency of the force curves is of 25 Hz) and for force 99

spectroscopy of 75 000 pN/s (acquisition frequency of the force curves is of 1.25 Hz). For 100

imaging and force spectroscopy, we used an AFM Nanowizard III (JPK Instruments, Berlin, 101

Germany). The cantilevers spring constants were determined by the thermal noise method

. For 102

all the results presented in this study, silicon nitride AFM tips were bare, expect in the case of 103

figure 4g (lower panel), where a functionalized AFM tip has been used.

104 105

AFM tips functionnalization 106

The functionalized tips were produced according to a french patent of the authors described later 107

in sensors and actuators

. Briefly, AFM tips were functionalized with dendrimers presenting

108

CHO functions able to covalently link with NH

functions of proteins. These dendritips were then 109

incubated with the lectin Concanavalin A (Sigma, L7647-100MG, 100µg/mL) for 1 hour, before 110

being used for force spectroscopy experiments.

111 112

Results analysis 113

All results were analyzed using the Data Processing software from JPK Instruments. The stiffness 114

values measured on cells were determined from the slope of the linear portion of the raw 115

deflections versus piezo displacement curves, according to:

116

with s the experimentally accessible slope of the compliance region reached for sufficient loading 117

forces. In this model, the experimental setup can be represented by two linear springs, one is the 118

AFM’s cantilever, and the other is the cell envelope exhibiting an effective spring constant. It is 119

then possible to calculate the effective spring constant kcell of the cell envelope from the 120

observed slope s of the force curve and the known spring constant k of the cantilever

. 121

122

Results 123

Candida albicans cells display localized adhesiveness 124

Thanks to our innovative method to immobilize cells into PDMS stamps

, and using 125

Quantitative Imaging

mode

, we were able to image and quantify the adhesive properties of 126

single C. albicans cells at the same time. Figure 1a shows a budding yeast cell; on the 127

corresponding adhesion image (Figure 1b), we can see that only the bud, and not the mother-cell, 128

presents adhesives patches. This original result is surprising as non-budding cells are highly 129

adhesive (see below). This result seems to indicate that the mother-cell cell wall changes during

130

the budding process. As for the cell in figure 1c, this cell displays two bud scars, a common 131

feature at the surface of yeast cells, which are not adhesive whereas the rest of the cell is. This 132

type of distribution of the adhesion on yeast cells has already been seen using 133

immunofluorescence with antibodies targeted against surface proteins of C. albicans. Coleman et 134

al. for example showed that the Als1 protein was expressed all over C. albicans cells, with the 135

exception of bud scars

. The comparison of our results to these data suggests then that the 136

adhesions probed by AFM might be due to surface proteins, such as Als1 in the case of the cell 137

presenting bud scars, but perhaps also others adhesins.

138 139

C. albicans cell wall adhesins are able to aggregate into nanodomains 140

As showed before

, the proteins expressed at the surface of C. albicans cell wall are 141

able to aggregate, and to form nanodomains. However, these nanodomains have not been yet 142

characterized at the nanoscale, nor were imaged at high resolution. In fact, these nanodomains 143

have specific adhesive properties that can be mapped using AFM in the Quantitative Imaging

144

mode. High resolution (256 pixels²) adhesive images are presented in Figure 2. It shows adhesive 145

nanodomains, at the surface of a living wild-type C. albicans cell. On the cell presented in this 146

figure (Figure 2a), the corresponding adhesion image shows very distinct adhesive nanodomains 147

that were probed with bare AFM tips. These nanodomains are homogeneously distributed all over 148

the cell here, which does not present any morphological features such as buds or bud scars. When 149

zooming into small areas on top of the cell (white squares on figure 2b), we could measure the 150

area of each nanodomain. On this cell and on another one showed in Figure 3a, 60 nanodomains 151

areas were measured; the values obtained plotted on figure 2e shows that nanodomains have an 152

average area of 0.09 ± 0.03 µm². This corresponds to an average diameter of 170 nm, which 153

confirms the nanoscale of these nanodomains. Some of the nanodomains are also higher than the

154

rest of the cell wall. When the whole cell is imaged, it is not visible; however, specific analysis of 155

the Figure 3d and the graphic 3h representing the topography of the cell surface revealed 156

nanodomains that had a different height compared to the rest of the cell. The cross-sections taken 157

along the blue line showed the height of a nanodomain of 20 nm. Once again, this confirmed the 158

nanoscale of the nanodomains at the surface of C. albicans.

159 160

Different nanodomains have different nanomechanical properties 161

Adhesion is measured as the rupture force recorded when retracting the tip from the surface, 162

when approaching and pulling with the tip on the cell wall, thus AFM makes it possible to 163

measure nanomechanical properties of living cells. Here we choose to use an analysis based on 164

the Hooke model which considers the coupled cantilever / cell wall as a spring. The stiffness 165

values measured on cells were determined from the slope of the linear portion of the raw 166

deflections versus piezo displacement curves, according to:

167

with s the experimentally accessible slope of the compliance region reached for sufficient loading 168

forces. Indeed, the most interesting result in this study is the correlation that can be directly made 169

between the adhesiveness of the nanodomain, and its stiffness. Nanodomains on figure 3e 170

(adhesion map) circled in red were found on the stiffness image (figure 3f) circled in black; they 171

correspond to the zones where the stiffness of the cell wall is increased, to 13.4 ± 0.3 nN/µm. As 172

for less adhesive nanodomains, they do not present any difference in stiffness from the rest of the 173

cell, and are 12.4 ± 0.2 nN/µm. The 3D-view of the adhesion, mapped with the stiffness (figure 174

3g) illustrates this clear correlation; the more adhesive the nanodomain is, the stiffer it is.

175

Another fascinating point is that for the more adhesive nanodomains, the retract force 176

curves present typical hydrophobic adhesions

, with adhesions occurring immediately when 177

the tips is retracted from the surface. Force curves from the other, less adhesive, nanodomains 178

presented retract adhesions resembling to proteins unfolding, occurring several nanometers after 179

the tip withdrawal. Therefore it seems that the nanodomains are of 2 different natures. There is a 180

class of nanodomains, hydrophobic, higher and stiff, and another class displaying proteins 181

unfolding properties, as soft as the rest of the cell wall. What is the molecular nature of these 2 182

types of nanodomains, and are they correlated?

183 184

Understanding the adhesive properties of the 2 nanodomains classes 185

To answer the previous question, we monitored the retract force curves recorded on the 186

nanodomains (Figure 4). We found the same correlation as in figure 3; the force curves recorded 187

on an adhesive nanodomains presented hydrophobic retract adhesions, whereas the force curves 188

recorded on a less adhesive nanodomain presented protein, unfolding like, profiles. In order to 189

determine the nature of these last unfoldings, we probed the surface of C. albicans cells with an 190

AFM tip functionalized with Concanavalin A (ConA), a protein that interacts with yeast 191

mannoproteins, such as surface adhesins. The resulting force curves (figure 4g) showed retract 192

adhesions displaying unfoldings of different lengths, but with a similar profile. We also observed 193

condensed spikes with adhesion forces between 0 and 50 pN. This value was consistent with 194

specific interactions between ConA and mannoproteins

. 195

In a previous study conducted in 2009

by Alsteens et al., adhesins (Als5) were unfolded 196

from the surface of live S. cerevisiae cells overexpressing this protein. The retract force curves 197

obtained in this study show high similarity with the ones we obtain here with functionalized AFM 198

tips, with the presence of serin-threonin rich segments (condensed pikes on figure 4g). We can

199

therefore, based on this comparison with the data of the literature, conclude that the less adhesive 200

nanodomains at the surface of live C. albicans are composed of free adhesins, and maybe of Als 201

proteins. However, since all adhesins (like Als, Hwp1, Eap1, Rbt1 etc) are mannoproteins, we 202

cannot, at this stage, make a statement on which adhesins are unfolded here.

203

As for the second type of nanodomains, the hydrophobic ones, our hypothesis is that they 204

are composed of the same proteins as the less adhesive ones. In fact, adhesins (like Als) display 205

amyloids sequences located on a domain of the protein called T, that enable them to change their 206

conformation

and to aggregate into amyloid nanodomains. And when this phenomena is started, 207

it propagates to the whole cell

. We therefore made the hypothesis that the adhesive nanodomains 208

are in fact amyloid nanodomains, made of Als proteins.

209 210

From adhesins to amyloid nanodomains: the role of Als proteins 211

To verify this hypothesis, and according to the literature on Als proteins, we synthesized a 212

peptide exhibiting the same sequence as the one of the T domain of the Als1/3/5 proteins. We 213

then put this peptide in the presence of the cells in order to trigger the amyloids formation. We 214

also synthesized the same peptide, but with a mutation on one amino acid (V326N peptide), in 215

order to obtain a peptide that inhibits the formation of the amyloid nanodomains

. Since Als3 is 216

only expressed on the surface of hyphae, we will only be able to generate or destroy the amyloid 217

formation of Als 1 and 5. The results presented in figure 5 showed a cell before and after adding 218

the mutated peptide. We can clearly see on these adhesion images the loss of general adhesion 219

and of two nanodomains at the center of the cell. It seems like the mutated peptide disrupted the 220

amyloids at the surface of the cell. And the other way around, when cells were incubated with the 221

amyloid forming peptide, we could observe the formation of the nanodomains at the surface of 222

the cells, as it is showed on the adhesion images on local areas on top of C. albicans cells in

223

figure 5c, d and e. These results allow us to conclude that the proteins at the origin of the 224

nanodomains are then mannoproteins, more specifically Als1 or 5, or both, that form amyloids.

225 226

Discussion 227

We show in this study that wild-type live C. albicans cells exhibit extraordinary adhesive 228

properties. In the case of budding cells, placed in acetate buffer at 25°C for 2h, we observed that 229

the mother cell is not adhesive and that only the bud presents adhesive nanodomains. On the 230

contrary, we show that non budding cells are covered by adhesive nanodomains, in the same 231

experimental conditions. This illustrates the amazing plasticity of this species

able to grow as 232

a commensal or as a pathogen

, in all the parts of the intestinal track, but also on the vaginal 233

mucosa, as unicellular budding cells or as filamentous hyphae. Moreover its cell wall is 234

permanently remodeled as a reaction to its environment (temperature, pH, dissolved O

, ions, 235

interacting surface/cells/bacteria) what makes it challenging to reproduce the experimental 236

conditions inducing a certain cell wall phenotype. We then demonstrate that the molecules at the 237

origin of these adhesions could aggregate into nanodomains, which can be probed at high 238

resolution using a suited AFM mode, QI

. These nanodomains are different in terms of level of 239

adhesiveness, which is a property directly correlated to their stiffness and to the hydrophobic 240

state or not of the molecule at the origin of these nanodomains. We then went further in the study, 241

using functionalized AFM tips, and were able to determine that the less adhesive nanodomains 242

were formed by mannoproteins that can interact specifically with Concanavalin A. These 243

mannoproteins are able to aggregate to form the adhesive nanodomains because they have 244

amyloid properties as we showed in figure5.

245

Amyloid aggregation is a primitive

and very stable

protein folding and a common 246

structural motif. It is a cross β-sheet quaternary structure that usually auto-aggregates as fibrils. It

247

has been, first, associated with neurodegenerative diseases like Alzheimer, Parkinson, or 248

Creutzfeldt-Jakob diseases. However it is more and more unclear if the amyloids lesions are the 249

cause or a consequence of the disease. Amyloid aggregates are now described as functional 250

proteins assembly and can be found from bacteria to humans

. In microorganisms, amyloid has 251

been described as a functional coat

. It constits in curli (E. coli), chaperons (Streptomyces) or 252

hydrophobins (Aspergillus etc); all of these proteins are implicated in adhesion to the host and in 253

the invasion, infection process. It is now well known that adhesins (and especially Als) of C.

254

albicans have amyloid-forming sequences

and that these proteins form domains involved in 255

cell aggregation or biofilm formation

. Nevertheless the characterization, structure and 256

properties of the amyloids adhesive nanodomains remain unclear.

257

In this work we measured for the first time the nanoscale size of amyloid domains 258

(average area of 0.09 µm

) at the surface of live C. albicans cells. The domains are of 2 different 259

classes. Some present the characteristic of individual proteins whereas the others are 260

hydrophobic, stiffer than the rest of the cell (13.4 ± 0.3 nN/µm compared to 12.4 ± 0.2 nN/µm), 261

and are slightly protruding. It means that there is a state modification from soluble proteins into 262

insoluble proteins, which is a characteristic of amyloid structures. This transformation is 263

dependent on the proteins concentration and can only occur when the protein density exceeds a 264

threshold. The roles of the two classes of domains are probably different. On one hand we could 265

hypothesize that the hydrophobic nanodomains were involved in the cell adhesion to abiotic 266

hydrophobic surfaces or to cell membrane as it is known that membrane binding is an inherent 267

property of amyloid aggregates

. Amyloid aggregation is also a way to store proteins, in a 268

limited space and to sort them when required. This has been demonstrated for hormones in 269

secretory granules

. Thus C. albicans may store some adhesins for the subsequent invasion 270

phases. On the other hand the protein like domains may be responsible for specific adhesion to

271

fibronectin and other extra cellular proteins of the matrix. It seems rational that several adhesins, 272

brought together, would be more efficient in a binding process than a single adhesion. This 273

finding has to be added to C. albicans plasticity

and participate to explain its remarkable 274

adaptation and pathogenicity.

275

However, there are still many things to explore on the cell wall of C. albicans, and future 276

work will be dedicated to exploring the changes appearing on the mother cell during the budding 277

process.

278 279

References 280

281

1. Gow NA, Hube B. Importance of the Candida albicans cell wall during commensalism and 282

infection. Curr Opin Microbiol. 2012;15:406–412.

283

2. Poulain D. Candida albicans , plasticity and pathogenesis. Crit Rev Microbiol. 2013:1–10.

284

3. Naglik JR, Moyes DL, Wächtler B, Hube B. Candida albicans interactions with epithelial 285

cells and mucosal immunity. Microbes Infect. 2011;13:963–976.

286

4. Hoyer LL. The ALS gene family of Candida albicans. Trends Microbiol. 2001;9:176–180.

287

5. Hoyer LL, Green CB, Oh S-H, Zhao X. Discovering the secrets of the Candida albicans 288

agglutinin-like sequence (ALS) gene family – a sticky pursuit. Med Mycol. 2008;46:1–15.

289

6. Ramsook CB, Tan C, Garcia MC, Fung R, Soybelman G, Henry R, et al. Yeast Cell 290

Adhesion Molecules Have Functional Amyloid-Forming Sequences. Eukaryot Cell.

291

2010;9:393 –404.

292

7. Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, et al. Atomic 293

structures of amyloid cross-β spines reveal varied steric zippers. Nature. 2007;447:453–457.

294

8. Garcia MC, Lee JT, Ramsook CB, Alsteens D, Dufrêne YF, Lipke PN. A Role for Amyloid 295

in Cell Aggregation and Biofilm Formation. PLoS ONE. 2011;6:e17632.

296

9. Rauceo JM, Gaur NK, Lee K-G, Edwards JE, Klotz SA, Lipke PN. Global Cell Surface 297

Conformational Shift Mediated by a Candida albicans Adhesin. Infect Immun.

298

2004;72:4948–4955.

299

10. Müller DJ, Dufrêne YF. Atomic force microscopy: a nanoscopic window on the cell 300

surface. Trends Cell Biol. 2011;21:461–469.

301

11. Alsteens D, Beaussart A, El-Kirat-Chatel S, Sullan RMA, Dufrêne YF. Atomic Force 302

Microscopy: A New Look at Pathogens. PLoS Pathog. 2013;9:e1003516.

303

12. Alsteens D, Garcia MC, Lipke PN, Dufrêne YF. Force-induced formation and propagation 304

of adhesion nanodomains in living fungal cells. Proc Natl Acad Sci. 2010;107:20744 – 305

20749.

306

13. Beaussart A, Alsteens D, El-Kirat-Chatel S, Lipke PN, Kucharíková S, Van Dijck P, et al.

307

Single-Molecule Imaging and Functional Analysis of Als Adhesins and Mannans during 308

Candida albicans Morphogenesis. ACS Nano. 2012;6:10950–10964.

309

14. Alsteens D, Dupres V, lotz SA, Gaur N , Lipke N, Dufr ne F. Unfolding Individual 310

Als5p Adhesion Proteins on Live Cells. ACS Nano. 2009;3:1677–1682.

311

15. Alsteens D, Ramsook CB, Lipke PN, Dufrêne YF. Unzipping a Functional Microbial 312

Amyloid. ACS Nano. 2012;6:7703–7711.

313

16. Chopinet L, Formosa C, Rols MP, Duval RE, Dague E. Imaging living cells surface and 314

quantifying its properties at high resolution using AFM in QI

mode. Micron. 2013;48:26–

315

33.

316

17. Dufrêne YF, Martínez-Martín D, Medalsy I, Alsteens D, Müller DJ. Multiparametric 317

imaging of biological systems by force-distance curve-based AFM. Nat Methods.

318

2013;10:847–854.

319

18. Dague E, Jauvert E, Laplatine L, Viallet B, Thibault C, Ressier L. Assembly of live micro- 320

organisms on microstructured PDMS stamps by convective/capillary deposition for AFM 321

bio-experiments. Nanotechnology. 2011;22.

322

19. Hutter JL, Bechhoefer J. Calibration of atomic-force microscope tips. Rev Sci Instrum.

323

1993;64:1868–1873.

324

20. Jauvert E, Dague E, Séverac M, Ressier L, Caminade A-M, Majoral J-P, et al. Probing 325

single molecule interactions by AFM using bio-functionalized dendritips. Sens Actuators B 326

Chem. 2012;168:436–441.

327

21. Arnoldi M, Fritz M, Bäuerlein E, Radmacher M, Sackmann M, Boulbitch A. Bacterial 328

turgor pressure can be leasured by atomic force microscopy. Phys Rev E. 1999;62:1034-1044 329

330

22. Coleman DA, Oh S-H, Zhao X, Hoyer LL. Heterogeneous distribution of Candida albicans 331

cell-surface antigens demonstrated with an Als1-specific monoclonal antibody.

332

Microbiology. 2010;156:3645–3659.

333

23. Dague E, Alsteens D, Latge JP, Verbelen C, Raze D, Baulard AR, et al. Chemical force 334

microscopy of single live cells. Nano Lett. 2007;7:3026–3030.

335

24. Alsteens D, Dague E, Rouxhet PG, Baulard AR, Dufrene YF. Direct measurement of 336

hydrophobic forces on cell surfaces using AFM. Langmuir. 2007;23:11977–11979.

337

25. Dague E, Alsteens D, Latgé J-P, Dufrêne YF. High-Resolution Cell Surface Dynamics of 338

Germinating Aspergillus fumigatus Conidia. Biophys J. 2008;94:656–660.

339

26. Alsteens D, Dupres V, Evoy KM, Wildling L, Gruber HJ, Dufrêne YF. Structure, cell wall 340

elasticity and polysaccharide properties of living yeast cells, as probed by AFM.

341

Nanotechnology. 2008;19.

342

27. Odds F. Candida and candidiosis. A review and bibliography. 2nd edition. London: Bailliere 343

Tindall; 1988.

344

28. Poulain D. Candida albicans , plasticity and pathogenesis. Crit Rev Microbiol. 2013:1–10.

345

29. Gow NA, Hube B. Importance of the Candida albicans cell wall during commensalism and 346

infection. Curr Opin Microbiol. 2012;15:406–412.

347

30. Gow NAR, van de Veerdonk FL, Brown AJP, Netea MG. Candida albicans morphogenesis 348

and host defence: discriminating invasion from colonization. Nat Rev Micro. 2012;10:112–

349

122.

350

31. Greenwald J, Riek R. Biology of Amyloid: Structure, Function, and Regulation. Structure.

351

2010;18:1244–1260.

352

32. Perczel A, Hudáky P, Pálfi VK. Dead-End Street of Protein Folding: Thermodynamic 353

Rationale of Amyloid Fibril Formation. J Am Chem Soc.;129:14959–14965.

354

33. Fowler DM, Koulov AV, Balch WE, Kelly JW. Functional amyloid--from bacteria to 355

humans. Trends Biochem Sci. 2007;32:217–224.

356

34. Gebbink MFBG, Claessen D, Bouma B, Dijkhuizen L, Wösten HAB. Amyloids — a 357

functional coat for microorganisms. Nat Rev Microbiol. 2005;3:333–341.

358

35. Otoo HN, Lee KG, Qiu W, Lipke PN. Candida albicans Als Adhesins Have Conserved 359

Amyloid-Forming Sequences. Eukaryot Cell. 2008;7:776–782.

360

36. Garcia MC, Lee JT, Ramsook CB, Alsteens D, Dufrêne YF, Lipke PN. A Role for Amyloid 361

in Cell Aggregation and Biofilm Formation. PLoS ONE. 2011;6:e17632.

362

37. Sparr E, Engel MFM, Sakharov DV, Sprong M, Jacobs J, de Kruijff B, et al. Islet amyloid 363

polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid 364

fibers. FEBS Lett. 2004;577:117–120.

365

38. Gellermann GP, Appel TR, Tannert A, Radestock A, Hortschansky P, Schroeckh V, et al.

366

Raft lipids as common components of human extracellular amyloid fibrils. Proc Natl Acad 367

Sci U S A. 2005;102:6297–6302.

368

39. Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, et al.

369

Functional Amyloids As Natural Storage of Peptide Hormones in Pituitary Secretory 370

Granules. Science. 2009;325:328–332.

371

40. Dannies PS. Concentrating hormones into secretory granules: layers of control. Mol Cell 372

Endocrinol. 2001;177:87–93.

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Figure Caption 375

Figure 1. Localization of the adhesive properties of C. albicans cells. (a) Height image (z- 376

range = 1.5 µm) of a budding C. albicans cell in a PDMS stamp, and (b) adhesion image 377

corresponding to the height image. On (a), MC stands for Mother cell, BC stands for Budding 378

cell, and the red dotted line represents the demarcation between the two different cells. (c) Height 379

image (z-range = 3.5 µm) of a single C. albicans cell exhibiting two bud scars, and (d) adhesion 380

image corresponding to the height image.

381 382

Figure 2. Imaging of the adhesive domains of C. albicans cells in acetate buffer, at 25°C for 383

2 hours. (a) Height image (z-range = 2.5 µm) of a single C. albicans cell in a 384

polydimethylsiloxane (PDMS) stamp, and (b) adhesion images corresponding to the height 385

images. (c, d) Adhesion images of small areas on top of the cell, represented by the white squares 386

in b. (e) distribution of the areas values of the domains in c and d.

387 388

Figure 3. Nanomechanics of the adhesive domains of C. albicans cells. (a) Height image (z- 389

range = 2.5 µm) of a C. albicans cell in a PDMS stamp, (b) corresponding adhesion image, and 390

(c) corresponding stiffness image. (d) Height image (z-range = 100 nm) of a small area on top of 391

the cell, represented by the white square on (a), (e) corresponding adhesion image and (f) 392

corresponding stiffness image. Note that the adhesive nanodomains circled in red on (e) are also

393

found on the stiffness image (black circles on f). (g) is a 3D-image of the adhesion mapped with 394

the stiffness. (h) cross-section taken along the blue line on (d), and (i), distribution of the stiffness 395

values corresponding to the yeast cell wall and the less adhesive domains (blue columns) or to the 396

most adhesive domains (yellow columns).

397 398

Figure 4. Adhesion force curves of C. albicans adhesive domains. (a) Height image (z-range = 399

4.0 µm) of a C. albicans cell in a PDMS stamp. (b) Adhesion image of a small area on top of the 400

cell, represented by the white square on (a). (c and d) representative force curves obtained on the 401

zones indicated by the arrows on (c). (e) Height image (z-range = 2.5 µm) of a C. albicans cell in 402

a PDMS stamp and (f) corresponding adhesion image recorded with a bare tip. (g) representative 403

force curves obtained in the zone delimited by a white square on (f) with the Con A tip.

404 405

Figure 5. Imaging of the adhesive domains of C. albicans cells treated with Als1, 3, 5p 406

amyloid disrupting peptide (V326N peptide) or Als1, 3, 5p amyloid forming peptide (Als 407

peptide). (a) Adhesion image of a single C. albicans cell in a PDMS stamp and (b) 408

corresponding adhesion image after adding V326N peptide. (c, d and e) Adhesion images of 409

small areas on top of a C. albicans cell after adding the Als peptide.

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